Inhibition of b-Adrenergic Receptors Induces a PersistentDeficit in Retrieval of a Cocaine-Associated Memory ProvidingProtection against Reinstatement

James M Otis1 and Devin Mueller*,1

1Department of Psychology, University of Wisconsin-Milwaukee, Milwaukee, WI, USA

Drug-seeking behavior is maintained by encounters with drug-associated cues. Preventing retrieval of drug-associated memories that

these cues provoke would therefore limit relapse susceptibility; however, little is known regarding the mechanisms of retrieval. Here, we

show that b-adrenergic receptor activation is necessary for the retrieval of a cocaine-associated memory. Using a conditioned place

preference (CPP) procedure, rats were conditioned to associate one chamber, but not another, with cocaine. When administered

before a CPP trial, propranolol, but not saline, prevented retrieval of a cocaine-associated CPP. In subsequent drug-free trials, rats

previously treated with propranolol continued to show a retrieval deficit, as no CPP was evident. This retrieval deficit was long lasting and

robust, as the CPP did not re-emerge during a test for spontaneous recovery 14 days later or reinstate following a priming injection of

cocaine. Moreover, the peripheral b-adrenergic receptor antagonist sotalol did not affect retrieval. Thus, retrieval of cocaine-associated

memories is mediated by norepinephrine acting at central b-adrenergic receptors. Our findings support the use of propranolol, a

commonly prescribed b-blocker, as an adjunct to exposure therapy for the treatment of addiction by preventing retrieval of drug-

associated memories during and long after treatment, and by providing protection against relapse.

Neuropsychopharmacology advance online publication, 4 May 2011; doi:10.1038/npp.2011.77

Keywords: noradrenergic b-receptor; norepinephrine; reinstatement; extinction learning; relapse; drug abuse

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INTRODUCTION

Drug addiction is a chronically relapsing disorder char-acterized by compulsive drug-seeking behavior, and ismaintained by encounters with drug-associated cues(Heather et al, 1991; Herman, 1974; O’Brien et al, 1990).Presentation of these cues can elicit craving in humans(Childress et al, 1986; Ehrman et al, 1992; Foltin and Haney,2000; O’Brien et al, 1977) and stimulate active drug seekingin rodents (Banna et al, 2010; Chauvet et al, 2009; Fuchset al, 2004; Mueller and Stewart, 2000). The associationsbetween these cues and the drug reinforcer are acquired,consolidated, and readily retrieved in the presence of thecues. Despite the pivotal role of drug-associated memoryretrieval in stimulating drug seeking and relapse, little isknown about the neurobiological mechanisms of retrieval.

Emerging evidence implicates the noradrenergic systemin memory retrieval in emotional learning paradigms (Sara,2000, 2009; van Stegeren, 2008). Stimulation of noradrener-

gic neurons in the locus coeruleus facilitates retrieval ofassociative memories (Devauges and Sara, 1991; Sara andDevauges, 1988), and the presentation of cues associatedwith emotional stimuli activates these neurons (Sterpenichet al, 2006) and provokes norepinephrine release (Cassenset al, 1980; Feenstra et al, 2001; Mingote et al, 2004). Miceconstitutively lacking dopamine b-hydroxylase (DBH), anessential enzyme in the synthesis of norepinephrine, areable to acquire contextual fear conditioning, but show adeficit in retrieval when tested on subsequent days(Murchison et al, 2004). Restoring noradrenergic functionin these mice before testing reversed the deficit. Further-more, systemic administration of propranolol, a b-adrener-gic receptor antagonist, also reduced or prevented memoryretrieval in mice and rats, indicating that retrieval of condi-tioned fear is dependent on these receptors (Murchisonet al, 2004; Rodriguez-Romaguera et al, 2009). Whetherretrieval of drug-associated memories is dependent onb-adrenergic receptors, however, remains unknown.

We assessed the necessity of b-adrenergic receptoractivation for the retrieval of cocaine-associated memoriesusing the conditioned place preference (CPP) procedure, ananimal model of drug seeking in which environmentalstimuli are associated with drug administration. First,we examined the effects of b-receptor blockade, using the

Received 6 November 2010; revised 4 April 2011; accepted 6 April2011

*Correspondence: Dr D Mueller, Department of Psychology, Uni-versity of Wisconsin-Milwaukee, Box 413, Milwaukee, WI 53201-0413,USA, Tel: + 1 414 229 6113, Fax: + 1 414 229 5219,E-mail: devinm@uwm.edu

Neuropsychopharmacology (2011), 1–9

& 2011 American College of Neuropsychopharmacology. All rights reserved 0893-133X/11 $32.00

www.neuropsychopharmacology.org

b-receptor antagonist propranolol, on initial CPP trials. Wethen determined whether the retrieval deficit induced bypropranolol is long lasting by examining subsequent CPPtrials in the absence of propranolol. We also determinedwhether the propranolol-induced retrieval deficit providesprotection against spontaneous recovery and cocaine-induced reinstatement. Our results provide clear evidencethat propranolol, a commonly prescribed b-blocker, couldbe useful in treating addiction by preventing drug-associated memories from being retrieved during and longafter treatment.

MATERIALS AND METHODS

Subjects

A total of 126 male long-Evans rats (Harlan Laboratories,Madison, WI) weighing 200–225 g were individually housedin clear plastic cages. Rats were maintained on a 14-h light/10 h dark cycle (lights on at 0700 hours) and had unlimitedaccess to both water and standard laboratory rat chow(Harlan Laboratories). Rats were weighed and handleddaily. All procedures were approved by the InstitutionalAnimal Care and Use Committee at the University ofWisconsin-Milwaukee in compliance with National Insti-tutes of Health guidelines.

Place Preference Apparatus

Behavioral testing and conditioning were conducted in athree-chamber apparatus in which two larger chambers(1300 � 900 � 11.500) were separated by a smaller chamber(600 � 700 � 11.500). One of the larger chambers had wire meshflooring with white walls, whereas the other had gold-gratedflooring with a black wall. The center chamber had PVCplastic floors. All floors were raised to 1.500, with removabletrays placed underneath. Removable partitions were used toisolate the rats within specific chambers during condition-ing. During the CPP trials, the partitions were removed toallow free access to the entire apparatus. Each of the largerchambers contained two infrared photobeams separated by300. If the beam furthest from the door was broken, the ratwas determined to be in the larger chamber. If only thebeam closest to the center chamber was broken, then the ratwas determined to be in the center chamber. In addition,total number of photobeam breaks could be recorded toquantify locomotor activity. During all phases of theexperiments, the room was kept in semidarkness.

Drugs

Cocaine HCl (National Institute on Drug Abuse) wasdissolved in sterile 0.9% saline at a concentration of10 mg/ml, and was administered intraperitoneally (i.p.) ata dose of 10 mg/kg for all experiments and 20 mg/kg inexperiment 1 only. ( + /�)-Propranolol HCl (Sigma-Aldrich,St Louis, MO) and sotalol HCl (Sigma-Aldrich) weredissolved in sterile 0.9% saline at a concentration of10 mg/ml, and were administered subcutaneously (s.c.) ata dose of 10 mg/kg. (�)-Propranolol HCl (Sigma-Aldrich)was dissolved in sterile 0.9% saline at a concentration of1 mg/ml, and was administered at a dose of 1 mg/kg, s.c.

Conditioning and Testing

Baseline preferences were determined by placing the rats inthe center chamber and allowing free access to all chambersfor 15 min. Time spent in each chamber was recorded.Overall, rats showed no preference for either of the largerconditioning chambers during this baseline trial, althoughless time was spent in the smaller center chamber. Analysisof variance (ANOVA) revealed a significant effect ofchamber (F2,250¼ 237.70, po0.001), and post hoc testsshowed that rats spent less time in the center chamberthan either of the larger chambers (po0.05) and anequivalent amount of time in the larger chambers(p40.05). Because of the absence of an initial preferencefor either conditioning chamber, we used an unbiasedprocedure in which rats were randomly assigned to receivecocaine in one or the other larger chamber. Followingbaseline testing, rats were conditioned to associate onechamber, but not another, with cocaine in a counter-balanced fashion over 8 days. Injections of saline or cocainewere given on alternating days immediately before 20-minconditioning sessions during which the rats were confinedto their respective chambers. Following four conditioningsessions in each chamber, the rats were subjected to a 2-daybreak, followed by daily CPP trials in which they wereplaced in the center chamber, with access to the entireapparatus for 15 min. A CPP was determined when ratsspent significantly more time in the previously cocaine-paired chamber than the saline-paired chamber.

Experimental Manipulations

To examine whether b-adrenergic receptor activation isnecessary for retrieval of cocaine-associated memories,we administered the non-selective b-adrenergic receptorantagonist ( + /�)-propranolol or saline 20 min beforecocaine-free CPP extinction trials. Rats were tested daily,receiving injections before the first trial only (experiment 1).In experiment 2, we investigated the possible nonspecificeffects of propranolol on CPP expression. First, to ensure thatthe effects of propranolol were specific to b-receptorblockade, we examined the effect of a low dose of the moreactive enantiomer (�)-propranolol (1 mg/kg, s.c.), withgreater specificity for b-receptors, on CPP retrieval. Ratswere tested daily, receiving (�)-propranolol or saline 20 minbefore the first CPP trial only. Second, we tested the possibleeffects of propranolol on reconsolidation. Rats were testeddaily, receiving ( + /�)-propranolol immediately before, asopposed to 20 min before, the first CPP trial, thereby allowingCPP retrieval during the first trial. Third, we examinedwhether propranolol itself induces a CPP or aversion.Following baseline testing, rats were conditioned to associateone chamber, but not another, with propranolol in acounterbalanced fashion over 2 days. Injections of ( + /�)-propranolol or saline were given on either day 20 min beforenormal conditioning sessions, during which the rats wereconfined to their respective chambers. Following condition-ing, the rats were subjected to a drug-free CPP extinctiontrial. Finally, the effect of propranolol on locomotor activitywas measured as photobeam breaks.

We next examined the effects of propranolol adminis-tered 20 min before the second trial only (experiment 3) or

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each of the second, third, and fourth trials (experiment 4).The multiple injection procedure was adopted to maximizethe retrieval deficit and model the use of a pharmacologicaladjunct to exposure therapy. Rats were then testedrepeatedly with daily drug-free CPP extinction trials. Inexperiment 4, rats were given an additional CPP trial 14days later to test for spontaneous recovery of the CPP.

In experiment 5, we examined whether propranololtreatment during initial CPP trials would prevent latercocaine-induced reinstatement. Rats received propranololor saline before each of the second, third, and fourth CPPtrials, and were then tested again in a drug-free CPP trial.To match the lack of CPP expressed by the propranololgroup after treatment, saline-treated rats were subjected to30-min drug-free CPP extinction trials (instead of thenormal 15-min CPP trials), with full access to the apparatusuntil no preference was observed (12 days). One dayfollowing the final CPP extinction trial for each treatmentgroup, rats were given a priming injection of cocaine(10 mg/kg, i.p.) 5 min before an additional CPP trial to testfor cocaine-induced reinstatement.

In experiment 6, we examined whether the effects ofpropranolol were due to b-receptor blockade within thecentral or peripheral nervous system. We injected either thenon-selective b-adrenergic receptor antagonist sotalol orsaline 20 min before the second, third, and fourth CPP trials.Sotalol does not cross the blood–brain barrier (Dahlof,1981), and has been shown to effectively decrease heart ratein rats to a similar extent as propranolol (Rodriguez-Romaguera et al, 2009).

Data Analysis

Drug-seeking behavior during single or across multiple CPPtrials was analyzed by comparing time spent in thepreviously cocaine-paired, saline-paired, and center cham-bers using a repeated measures ANOVA. Following asignificant effect of chamber, Tukey’s Honestly SignificantDifference post hoc tests were used to compare the amountof time spent in the previously cocaine-paired vs saline-paired chambers for single or across multiple CPP trials. Inno cases did animals spend significantly more time in thecenter chamber than in the cocaine-paired chamber(p40.05). Locomotor activity was measured as the numberof photobeam breaks during the CPP trials, and wasanalyzed using an independent sample t-test.

RESULTS

Propranolol Prevents Retrieval of a CPP whenAdministered Before an Initial Test

We first examined the necessity of b-adrenergic receptoractivation for the retrieval of a cocaine-associated CPP. Inexperiment 1, rats were pretested, conditioned with either10 or 20 mg/kg of cocaine, and subjected to daily CPP trials,with administration of the b-adrenergic receptor antagonistpropranolol (10 mg/kg) or saline (n¼ 6–8 per group) 20 minbefore the first CPP trial. Rats injected with salinedemonstrated a CPP for the previously cocaine-pairedchamber during this trial, but rats injected with propranololdid not (see Figure 1). ANOVA revealed a significant effect

of chamber for saline-treated rats conditioned with eitherdose of cocaine on the first trial (10 mg/kg cocaine,F2,14¼ 8.51, po0.01; 20 mg/kg cocaine, F2,14¼ 21.35,po0.001), and post hoc analyses confirmed that signifi-cantly more time was spent in the previously cocaine-pairedchamber than the saline-paired chamber (po0.01). Propra-nolol-treated rats conditioned with either dose of cocaine,however, spent an equal amount of time in the previouslycocaine-paired chamber and saline-paired chamber (effectof chamber: 10 mg/kg cocaine, F2,14¼ 3.73, p40.05, post hocp40.05; 20 mg/kg cocaine, F2,10¼ 11.83, po0.01, post hocp40.05), indicating a lack of a CPP. Across all subsequentCPP trials (ie, trials 2–6), saline-treated rats conditionedwith either dose of cocaine continued to spend significantlymore time in the cocaine-paired chamber than the saline-paired chamber overall (effect of chamber: 10 mg/kgcocaine, F2,14¼ 10.82, p¼ 0.001, post hoc po0.001; 20 mg/kgcocaine, F2,14¼ 9.04, po0.01, post hoc po0.01), whereaspropranolol-treated rats spent an equivalent amount of timein all chambers (10 mg/kg cocaine, F2,14¼ 2.78, p40.05;20 mg/kg cocaine, F2,10¼ 1.71, p40.05). Thus, a singletreatment of propranolol disrupted initial and subsequentretrieval of a CPP.

At high doses, propranolol has been shown to have effectsin addition to b-receptor blockade, including serotonergicreceptor blockade (Alexander and Wood, 1987) and proteinkinase C inhibition (Sozzani et al, 1992). Thus, inexperiment 2, we examined the effect of a low dose of(�)-propranolol, the more active enantiomer that hasgreater specificity for b-receptors (Dawson, 1986), on CPPexpression. Rats were pretested, conditioned, and subjected

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Figure 1 Propranolol disrupts retrieval of a cocaine-associated condi-tioned place preference (CPP). Following conditioning with (a) 10 mg/kg or(b) 20 mg/kg doses of cocaine, propranolol but not saline injections(arrows) prevented rats from expressing a CPP for the previously cocaine-paired chamber over the previously saline-paired chamber during the firstCPP trial. Propranolol- but not saline-treated rats continued to show noCPP during subsequent propranolol-free trials. ***po0.001, **po0.01,and *po0.05.

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to daily CPP trials, with administration of (�)-propranolol(1 mg/kg; n¼ 8) or saline (n¼ 8) 20 min before the first CPPtrial. Rats injected with saline demonstrated a CPP for thepreviously cocaine-paired chamber during this trial, butrats injected with (�)-propranolol did not (see Figure 2a).ANOVA revealed that saline-treated rats spent significantlymore time in the previously cocaine-paired chamber duringthis trial (effect of chamber: F2,14¼ 23.18, po0.001, post hocpo0.001), whereas propranolol-treated rats spent anequivalent amount of time in the cocaine- and saline-pairedchambers (effect of chamber F2,14¼ 8.99, po0.05, post hocp40.05). During subsequent propranolol-free trials (ie,trials 2–5), saline-treated rats continued to spend signifi-cantly more time in the cocaine-paired chamber overall(effect of chamber: F2,14¼ 13.14, po0.001, post hocpo0.01), whereas propranolol-treated rats spent an equiva-lent amount of time in the cocaine-paired and saline-pairedchambers (F2,14¼ 39.46, po0.001, post hoc p40.05). Thus,(�)-propranolol disrupted the initial and subsequentretrieval of a CPP, indicating that the effects of propranololon CPP retrieval are specific to b-receptor blockade.

Propranolol has been demonstrated to impair reconsoli-dation of a cocaine-associated memory when administeredafter a retrieval trial (Bernardi et al, 2006; Fricks-Gleasonand Marshall, 2008), and therefore reconsolidation blockadecould explain the persistent retrieval deficit observed in our

study. To test this, we administered propranolol immedi-ately before, as opposed to 20 min before, an initial CPPtrial. Rats injected with propranolol (n¼ 7) demonstrated aCPP for the previously cocaine-paired chamber during thistrial and during subsequent drug-free trials (see Figure 2b).ANOVA revealed that propranolol-treated rats spentsignificantly more time in the previously cocaine-pairedchamber during this trial (effect of chamber: F2,12¼ 15.01,po0.001, post hoc po0.001). During subsequent drug-freetrials, propranolol-treated rats continued to spend signifi-cantly more time in the previously cocaine-paired chamber(effect of chamber: F2,12¼ 7.89, p¼ 0.01, post hoc po0.05).Thus, a single propranolol treatment immediately before aninitial CPP trial did not disrupt subsequent CPP expression.Taken together, these results indicate that propranololinduces a persistent CPP retrieval deficit independently ofreconsolidation blockade.

We next examined whether propranolol itself induced anaffective state or influenced locomotor activity. We condi-tioned rats (n¼ 11) to associate one chamber, but notanother, with propranolol. Propranolol treatment had noeffect on time spent in the conditioning chambers during asubsequent drug-free CPP trial (see Figure 2c). ANOVArevealed a significant effect of chamber during the CPP trial(F2,20¼ 18.58, po0.001), but post hoc analyses indicatedthat an equivalent amount of time was spent in thepreviously propranolol- and saline-paired chambers(p40.05). Thus, propranolol did not generate an affectivestate sufficient to induce a CPP or aversion, consistent withprevious findings (Milton et al, 2008). We also evaluated theeffects of propranolol on locomotor activity. Rats injectedwith saline (n¼ 8) or propranolol (n¼ 8) before an initialCPP trial had similar levels of locomotor activity during thetrial (t14¼ 1.16, p¼ 0.25; see Figure 2d). Furthermore, ratsinjected with saline (n¼ 8) or (�)-propranolol (n¼ 8)before an initial CPP trial had similar levels of locomotoractivity during the trial (beam breaks: saline, 218.8±11.2;(�)-propranolol, 228.3±10.3; t14¼ 0.62, p40.05; data notshown). Thus, retrieval deficits induced by propranolol arenot due to nonspecific motor impairments.

Propranolol Prevents Retrieval after a CPP has Alreadybeen Expressed

To test whether retrieval could be disrupted after a CPP hadbeen previously expressed, in experiment 3, we examinedthe effects of propranolol on retrieval during the secondCPP trial. Following conditioning, all rats expressed a CPPfor the previously cocaine-paired chamber during the firsttrial. Injections of propranolol (n¼ 11), but not saline(n¼ 15), before the second trial impaired expression of theCPP on that trial and all subsequent trials (see Figure 3).ANOVA revealed an effect of chamber for both groupsduring the first trial (saline F2,28¼ 24.91, po0.001; propra-nolol, F2,20¼ 10.10, po0.001), and post hoc analysesconfirmed that both groups spent significantly more timein the cocaine-paired chamber than the saline-pairedchamber (po0.001). Following injections of propranololor saline before the second trial, however, only the saline-treated rats spent significantly more time in the previouslycocaine-paired chamber (effect of chamber: F2,28¼ 19.71,po0.001, post hoc po0.001), whereas propranolol-treated

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Figure 2 Propranolol-induced conditioned place preference (CPP)retrieval disruption is not due to nonspecific effects of propranolol orreconsolidation blockade. (a) Following conditioning, the more activeenantiomer of propranolol, (�)-propranolol but not saline injections(arrows) prevented rats from expressing a CPP for the previously cocaine-paired chamber over the previously saline-paired chamber during the firstand subsequent CPP trials. (b) Propranolol injection immediately before thefirst CPP trial did not prevent CPP expression during this trial orsubsequent trials. (c) Following conditioning with propranolol or saline, ratsspent an equivalent amount of time in the previously propranolol-pairedand saline-paired chambers. (d) Propranolol had no effect on locomotoractivity, as measured by photobeam breaks, when injected before an initialCPP trial. Prop, propranolol. ***po0.001, **po0.01, and *po0.05.

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rats spent an equivalent amount of time in all chambers(F2,20¼ 2.68, p40.05). During subsequent propranolol-freetrials (ie, trials 3–6), saline-treated rats continued to spendsignificantly more time in the cocaine-paired chamberoverall (effect of chamber: F2,20¼ 5.90, po0.01, post hocpo0.001), whereas propranolol-treated rats spent anequivalent amount of time in all chambers (F2,12¼ 0.86,p40.05). Thus, propranolol disrupted the retrieval of apreviously expressed CPP.

Repeated Propranolol Treatment Results in aLong-Lasting Retrieval Deficit

The passage of time is sufficient to induce spontaneousrecovery of associative memories (Brooks and Bouton, 1993;Rescorla, 2004). Thus, we examined whether the proprano-lol-induced retrieval deficit would persist when rats weretested 14 days after treatment (experiment 4). Followingconditioning, all rats expressed a CPP for the previouslycocaine-paired chamber during the first trial. Propranololtreatment before each of the second, third, and fourth trialsdisrupted this CPP during these and a subsequent drug-freetrial (see Figure 4a). ANOVA revealed that both treatmentgroups (saline, n¼ 6; propranolol, n¼ 7) spent significantlymore time in the cocaine-paired chamber on the first trial(saline F2,12¼ 20.67, po0.001; propranolol, F2,10¼ 10.86,

po0.01, post hoc po0.05). During subsequent trials (ie,trials 2–5), saline-treated rats continued to spend signifi-cantly more time in the previously cocaine-paired chamberoverall (effect of chamber: F2,12¼ 8.48, po0.01, post hocpo0.001), whereas propranolol-treated rats spent anequivalent amount of time in all chambers (F2,10¼ 3.34,p40.05). Thus, repeated propranolol administrationblocked the retrieval of a CPP during and followingtreatment. To determine whether this effect was resistantto spontaneous recovery, the rats were subjected to a CPPtrial 14 days later. Propranolol-treated rats did not express aCPP during this trial as compared with saline-treated rats(see Figure 4b). ANOVA revealed that saline-treated ratsspent significantly more time in the previously cocaine-paired chamber (effect of chamber: F2,12¼ 5.59, po0.05,post hoc po0.05), whereas propranolol-treated rats spent anequivalent amount of time in all chambers (F2,10¼ 0.81,p40.05). Thus, repeated propranolol treatment results in along-lasting retrieval deficit for a previously expressed CPP.

Propranolol Protects against Cocaine-InducedReinstatement

A priming injection of cocaine has been shown to reliablyinduce reinstatement of a CPP after extinction (Kelley et al,2007; Mantsch et al, 2010; Mueller and Stewart, 2000;Rodriguez-Arias et al, 2009). Behaviorally, both propranololtreatment and extinction training abolish the expression ofa CPP; hence, in experiment 5, we determined whether theretrieval deficit induced by propranolol would preventreinstatement of a CPP to a priming injection of cocaine.Rats were injected with saline (n¼ 11) or propranolol(n¼ 9) before the second, third, and fourth CPP trials, withsaline-treated rats receiving an additional eight CPP trials oflonger duration (30 min) to ensure extinction of the CPP. Tocompare the final extinction trial of saline-treated rats withthat of propranolol-treated rats, we analyzed the first 15 minof the final CPP extinction trial. Rats in both groups showedno chamber preference by the final trial. One day later, ratswere injected with cocaine (10 mg/kg, i.p.) 5 min before aCPP trial to test for reinstatement of the CPP. Previouspropranolol treatment prevented cocaine-induced reinstate-ment of a CPP as compared with previous saline treatment(see Figure 5). ANOVA revealed that both groups spent anequivalent amount of time in all chambers during the final

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Figure 3 Propranolol disrupts retrieval of an established conditionedplace preference (CPP). All rats expressed a CPP for the previouslycocaine-paired chamber over the previously saline-paired chamber duringthe first CPP trial. Propranolol but not saline injections (arrows) preventedrats from expressing a CPP during the second CPP trial. Propranolol- butnot saline-treated rats continued to show no CPP during subsequentpropranolol-free trials. ***po0.001 and *po0.05.

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Figure 4 Repeated propranolol treatment causes a long-lasting disruption in the retrieval of a conditioned place preference (CPP). (a) All rats expressed aCPP for the previously cocaine-paired chamber over the previously saline-paired chamber during the first CPP trial. Propranolol but not saline injections(arrows) before the second, third, and fourth CPP trials prevented the rats from expressing a CPP during those trials and a subsequent propranolol-free CPPtrial. (b) Propranolol but not saline treatment resulted in a lack of expression of a CPP during a CPP trial 14 days later. Prop, propranolol. ***po0.001,**po0.01, and *po0.05.

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CPP trial (saline F2,20¼ 0.57, p40.05; propranolol,F2,16¼ 0.68, p40.05). During the propranolol-free cocaine-primed reinstatement trial, however, saline-treated ratsspent significantly more time in the previously cocaine-paired chamber (effect of chamber: F2,20¼ 4.54, po0.05,post hoc po0.01), whereas propranolol-treated rats spent anequivalent amount of time in all chambers (F2,16¼ 0.07,p40.05). Thus, only saline-treated rats expressed cocaine-induced reinstatement of a CPP, indicating that disruptingretrieval of a CPP with propranolol subsequently preventscocaine-induced reinstatement in the absence of furtherpropranolol treatment.

Propranolol-Induced Retrieval Deficits are MediatedCentrally

Propranolol acts both centrally and peripherally (Streetet al, 1979), and therefore retrieval deficits could bemediated by feedback from the peripheral nervous system.To address this, in experiment 6, we administered sotalol, ab-adrenergic receptor antagonist that does not cross theblood–brain barrier (Dahlof, 1981). Following conditioning,all rats expressed a CPP for the previously cocaine-pairedchamber during the first trial. Rats were then injected withsaline (n¼ 6) or sotalol (10 mg/kg, s.c.; n¼ 9) before thesecond, third, and fourth CPP trials (see Figure 6). ANOVArevealed that both groups spent significantly more time inthe previously cocaine-paired chamber during the first trial(saline, F2,10¼ 18.67, po0.001; sotalol F2,16¼ 38.64,po0.001) and during all trials following treatment (saline,F2,10¼ 8.59, po0.01; sotalol F2,16¼ 6.31, po0.01). Post hocanalyses confirmed that saline- and sotalol-treated ratsspent significantly more time in the cocaine-paired chamberthan the saline-paired chamber across all trials (po0.01).Thus, b-adrenergic receptor blockade in the periphery alonedoes not affect retrieval of a CPP, indicating that the effectsof propranolol must be mediated centrally.

DISCUSSION

We investigated the effects of the b-adrenergic receptorantagonist propranolol on the retrieval of a cocaine-

associated CPP. Propranolol disrupted initial retrieval ofthe CPP and prevented subsequent retrieval in the absenceof further propranolol treatment. This retrieval deficit waslong lasting, perhaps permanent, as the CPP never re-emerged in subsequent trials and provided protectionagainst cocaine-induced reinstatement. The effects ofpropranolol on memory retrieval were mediated centrally,as the peripheral b-receptor antagonist sotalol had no effect.Taken together, these data demonstrate for the first timethat b-adrenergic receptor activation is necessary for theretrieval of a CPP, and preventing this activation results in along-lasting, state-independent deficit in retrieval of acocaine-associated memory.

Our finding that propranolol disrupts the retrieval of acocaine-associated memory agrees with previous investiga-tions. b-Adrenergic signaling has a pivotal role in retrievalof context-conditioned fear (Murchison et al, 2004; Ouyanget al, 2008) and the expression of cue-conditioned fear(Rodriguez-Romaguera et al, 2009), both of which aredisrupted by propranolol. Propranolol prevents stress-induced reinstatement of drug seeking (Mantsch et al,2010) and context-induced reinstatement of cue-condi-tioned fear (Morris et al, 2005), which could be attributed tomemory retrieval blockade. In humans, declarative memoryenhancement of emotional words is persistently disruptedby a single propranolol treatment (Kroes et al, 2010).Similarly, in abstinent heroin addicts, propranolol disruptsstress-enhanced drug-associated memory retrieval (Zhaoet al, 2010). In light of our results, these findings may bedue to memory retrieval blockade rather than simply areduction in stress. In our hands, propranolol treatmentresulted in a permanent retrieval deficit, as the CPP did notre-emerge after 14 days in a test for spontaneous recoveryor after a priming injection of cocaine. This latter effect isremarkable, as cocaine has been shown to reliably inducereinstatement of a CPP after extinction (Kelley et al, 2007;Mantsch et al, 2010; Mueller and Stewart, 2000; Rodriguez-Arias et al, 2009). Because the passage of time and cocaineitself did not reinstate the CPP, we conclude that retrieval ofthe original association between cocaine and the condition-ing context was disrupted.

Our finding that b-adrenergic signaling is necessary forCPP retrieval is supported by studies using mice lacking

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Figure 5 Propranolol treatment prevents subsequent cocaine-inducedreinstatement of a conditioned place preference (CPP). Saline- andpropranolol-treated rats showed no CPP for the previously cocaine-pairedchamber over the previously saline-paired chamber on the final extinctiontrials. The following day, saline- but not propranolol-treated rats expresseda cocaine-induced reinstatement of the CPP. Sal, saline; prop, propranolol.**po0.01.

Tim

e in

Cha

mbe

r (s

ec)

Trial

Saline Sotalol

Trial

Figure 6 Peripheral b-adrenergic receptor antagonist sotalol does notimpair retrieval of a conditioned place preference (CPP). All rats expresseda CPP for the previously cocaine-paired chamber over the previouslysaline-paired chamber during the first CPP trial. Peripheral b-adrenergicreceptor antagonist sotalol and saline injections (arrows) before thesecond, third, and fourth CPP trials did not alter expression of a CPP acrosstrials. ***po0.001, **po0.01, and *po0.05.

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DBH, an essential enzyme for the synthesis of norepinephr-ine from dopamine. Although DBH-knockout mice wereable to express a CPP to one of three conditioning doses ofcocaine in one study (Schank et al, 2006), this was not truein another (Jasmin et al, 2006). In addition, DBH-knockoutmice were unable to express a CPP after conditioning withfive different doses of morphine (Olson et al, 2006).Importantly, both cocaine- and morphine-induced CPPexpression was rescued in DBH-knockout mice followingnorepinephrine restoration by administration of DOPSduring conditioning (Jasmin et al, 2006; Olson et al,2006). The authors concluded that norepinephrine mediatesthe rewarding properties of addictive drugs. However,because norepinephrine was restored in both studies 24 hbefore the CPP trial, detectable levels of norepinephrinewould have remained in the brain (Thomas et al, 1998).Taken together with the results of the current study, theseresults demonstrate a critical role of noradrenergic signal-ing in retrieval of a drug-induced CPP. The necessity ofnorepinephrine for retrieval may be specific to drug-associated memories, as DBH-knockout mice are able toexpress a food-induced CPP (Jasmin et al, 2006; Olson et al,2006). Whether norepinephrine is necessary for retrieval ofdrug-associated memories in a self-administration para-digm remains to be determined.

Recent investigations have demonstrated a role forb-adrenergic signaling in the reconsolidation of drug-associated memories. Post-retrieval propranolol treatmentdisrupts subsequent expression of both cocaine- andmorphine-associated CPP memories (Bernardi et al, 2006;Fricks-Gleason and Marshall, 2008; Robinson and Franklin,2007), an effect attributable to reconsolidation blockaderather than facilitated extinction learning (Fricks-Gleasonand Marshall, 2008). In the present study, we found thatpre-retrieval propranolol treatment blocked the initialretrieval of a CPP memory. This cannot be explained byimpaired reconsolidation, as memory retrieval is notdisrupted until several hours following reconsolidationblockade (Lewis, 1979; Lubin and Sweatt, 2007; Nader andHardt, 2009; Nader et al, 2000). Moreover, initialmemory retrieval is required for reconsolidation to occur(Nader and Hardt, 2009; Nader et al, 2000). Our resultsshow that the initial retrieval was blocked by propranololindependently of CPP expression deficits, as propranololhad no effect on locomotor activity and did not induce aCPP or aversion on its own. We also showed that allowingmemory retrieval by administering propranolol immedi-ately rather than 20 min before CPP testing spared CPPexpression during subsequent trials. Thus, our results arecongruent with a permanent impairment in memoryretrieval rather than disrupted memory reconsolidation.This is in agreement with studies in other learning andmemory paradigms, demonstrating that manipulations caninduce persistent memory retrieval deficits independentlyof blocking memory reconsolidation (Kim and Fanselow,1992; Kroes et al, 2010; Miller and Springer, 1972; Sara,1973, 2000).

Another interpretation of our results is that propranololfacilitated the extinction of a CPP. This is unlikely, however,because of the complete absence of a CPP followingpropranolol administration before a CPP trial. Furthermore,it is well accepted that norepinephrine facilitates, rather

than impedes, memory formation through b-receptoractivation (McGaugh, 2000). Finally, research has demon-strated that propranolol impairs extinction learning(for review see Mueller and Cahill, 2010) in both aversive(Do-Monte et al, 2010; Merlo and Izquierdo, 1967; Muelleret al, 2008; Ouyang and Thomas, 2005) and appetitiveparadigms (LaLumiere et al, 2010).

Our findings indicate that the long lasting, perhapspermanent, disruption of retrieval of cocaine-associatedmemories that results from b-adrenergic receptor blockadeis mediated centrally, implicating central norepinephrinerelease in memory retrieval. The locus or loci at which thesereceptors mediate retrieval, however, remain to be deter-mined. Evidence indicates that b-receptors in the dorsalhippocampus mediate retrieval of other contextual mem-ories, although the b-receptor dependence is limited to3 days following conditioning (Murchison et al, 2004). Inexperiments 2, 3, and 4, propranolol was administered 4days following the last of eight conditioning trials, andinduced a long-term deficit in CPP retrieval. Thus, theeffect of propranolol on CPP retrieval may be independentof the hippocampus and is unlikely to have the sametime limitations as the retrieval of other hippocampus-dependent contextual memories. Whether remote CPPmemories (weeks or months after conditioning) areamenable to disruption by propranolol, however, remainsto be determined.

Although the noradrenergic mechanisms underlyingretrieval are poorly understood, activation of locus coer-uleus noradrenergic neurons likely mediates retrieval. Theseneurons show a graded response to stimuli, respondinggreatest to particularly salient and relevant stimuli (Sara,2009). Thus, the attribution of salience to stimuli followinglearning suggests that conditioned stimuli are particularlycapable of evoking noradrenergic responses. In fact, locuscoeruleus neurons respond vigorously to changes instimulus-reinforcement contingencies, and this neuralresponse precedes changes in behavioral responses(Aston-Jones et al, 1997; Bouret and Sara, 2004; Sara andSegal, 1991). Furthermore, recording studies in freelymoving rats have shown that locus coeruleus neurons firein response to salient stimuli on the first few presentationseven in the absence of the unconditioned stimulus, but theneural response declines when the stimulus no longerevokes a conditioned response (Bouret and Sara, 2004; Saraand Segal, 1991). Direct stimulation of locus coeruleusneurons facilitates memory retrieval (Devauges and Sara,1991; Sara and Devauges, 1988), supporting the notion thatstimulus-evoked norepinephrine release initiates memoryretrieval. As a result, disruption of noradrenergic signalingby propranolol prevents retrieval (Murchison et al, 2004;Ouyang et al, 2008; present findings) and may render drug-associated stimuli less salient. This effect could be due to adeficit in attention, such that rats are unaware of thesalience previously attributed to the drug-associatedchamber. Thus, subsequent presentations of those stimulino longer evoke norepinephrine release, resulting in apermanent deficit in memory retrieval. Accordingly, nora-drenergic stimulation (via noradrenergic agonists or directlocus coeruleus stimulation) may induce memory retrieval,as demonstrated in other paradigms (Devauges and Sara,1991; Sara and Devauges, 1988, 1989). These data suggest

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that the original memory remains intact, but the retrievalmechanism has been disrupted. Thus, reconditioningpreviously propranolol-treated rats might be expected tostrengthen the original CPP memory and re-engage retrievalmechanisms that were previously disrupted.

The presentation of drug-associated stimuli promotesdrug seeking and relapse in addicts (Ehrman et al, 1992;Foltin and Haney, 2000; Heather et al, 1991; O’Brien et al,1990; O’Brien et al, 1977). Thus, pharmacological manip-ulations that disrupt retrieval of long-term memories offer aparticularly efficacious avenue for therapies targeting theelimination of these drug-associated memories. Rehabilita-tion programs utilizing exposure therapy, in which addictsare presented with drug-associated stimuli in the absence ofthe drug, have had limited success without the use ofpharmacological adjuncts (Conklin and Tiffany, 2002;O’Brien et al, 1990). There are, however, no FDA-approvedpharmacological adjuncts to exposure therapy for thetreatment of addiction. Our findings suggest thatpropranolol would be a useful adjuvant during exposuretherapy to permanently disrupt stimulus-induced drugseeking and prevent drug-induced relapse long aftertreatment cessation.

ACKNOWLEDGEMENTS

This research was supported by DA027870 and a grant fromthe University of Wisconsin-Milwaukee Research GrowthInitiative to DM. We thank Kidane Dashew and Daniel Yangfor technical assistance, Dr Robert Twining for insight intoexperimental design, and Patrick Reilly for design andconstruction of the CPP boxes.

DISCLOSURE

The authors declare no conflict of interest.

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